This invention relates to a method for disinfecting or sterilizing objects in liquid phase with improved chemosterilizer compositions. The method object of our invention is based upon the synergistic sporicidal effects observed when using relatively moderate temperatures combined or not combined with ultrasonic irradiation in specially formulated sporicidal compositions. The latter are based upon active combinations of glutaraldehyde with nonionic surfactants such as ethoxylates or isomeric linear alcohols (C.sub.11 to C.sub.15) or anionic alkyl aryl sulfonates.
Through a proper choice of temperatures, acoustic energy density and chemical composition, the method object of the present invention enables reducing from hours to minutes the time requirements for surface disinfection or sterilization of heat sensitive materials.
Low temperature surface sterilization in liquid phase has been limited in the past to the use of two chemosterilizer agents: formaldehyde and alkaline glutaraldehyde solutions. This limited choice indeed contrasts with the large number of chemical bactericides available (Quarternary Ammonium compounds, chlorine containing compounds, Iodophores, Amphoteric compounds, etc.) when one does not require sporicidal action.
Formaldehyde is one of the oldest chemosterilizers employed for the destruction of spores, and, although 1 to 2% solutions have been used, a relatively long period of time (up to 20 hours) is required to destroy Bacillus subtilis var. niger spores. A somewhat shorter time is needed if one uses higher concentrations of formaldehyde (around 8 per cent) in isopropyl alcohol. This solution, called Formalin has several drawbacks. The irritating fumes of formaldehyde limit its usefulness, and its toxicity for tissue requires that disinfected materials be thoroughly rinsed with sterile water before use.
Alkalinized glutaraldeyde solutions known commercially under the trade name CIDEX are the only widely used for practical applications today. They consist of 2% aqueous glutaraldehyde solution buffered by suitable alkalinating agents (generally 0.3% sodium bicarbonate) to pH of 7.5 to 8.5. In the acid state at room temperature the glutaraldehyde solution is stable for long periods of time when stored in a closed container. However, when rendered alkaline, the glutaraldehyde gradually undergoes polymerization and loses its activity. Above pH 9 the polymerization proceeds very rapidly. In the 7.5 to 8.5 pH range polymerization is slower, but it is acknowledged by the manufacturer himself that sporicidal activity disappears after two weeks. (ARBROOK, Bulletin JR 8016, 1968)
Even when using a fresh solution of 2% buffered glutaraldehyde, the time needed at room temperature to achieve complete sterilization of Bacillus subtilis with the AOAC Pennycylinder method is said to be comprised between 3 and 10 hours according to spore dryness.
The impossibility to store the sporicidal solution over extended periods of time, the need to buffer each time before use and the long contact time required (several hours) to achieve sterility made us develop the method and new sporicidal composition objects of the present invention.
As hereabove stated, Alkalinized Glutaraldehyde has been widely used as a chemical sterilizing agent since its antimicrobial characteristics were first described in the U.S. Pat. No. 3,016,328 (1962). R. E. Pepper and E. R. Lieberman were the first to point out in the abovementioned patent that aqueous glutaraldehyde solutions were mildly acid and in this state they stressed that they did not exhibit sporicidal characteristics. Only when the solution was buffered by suitable alkalinating agents to a pH of 7.5 to 8.5, did the solution become antimicrobially active. (see American Journal of Hospital Pharmacy 20: 458-465, Sept. 1963) This point was emphasized in the U.S. Pat. No. 3,016,328 (1962) which stated (page 1, column 2, line 34) that the invention resided in the discovery that a saturated dialdehyde containing 2 to 6 carbons does, in fact, have sporicidal activity when it is combined with a lower alkanol and an alkalinating agent.
More recently G. Sierra in Canada (Can. Patent No. 865,913, March 1971) showed that the conclusions of R. E. Pepper and E. R. Lieberman were only valid in the temperature range (22.degree.-23.degree.C) indicated by these authors in their U.S. patent. The Sierra's Canadian patent indicates that strong sporicidal activity is exhibited by acid non-buffered non-alkalinized glutaraldehyde solutions when operating at temperatures higher (generally around 45.degree.C) than those mentioned in R. E. Pepper's patent. This observation was confirmed in our own experiments. Moreover, we found, and this is one of the objects of the present invention, that with the proper combination of acid glutaraldehyde with certain nonionic or anionic surfactants at temperatures greater than 15.degree.C but specially above 45.degree.C higher sporicidal activities than those mentioned in G. Sierra's patent can be achieved.
An increase in bactericidal and sporicidal activity through the combined use of glutaraldehyde (both acid and alkaline) with surfactants had been previously disclosed by A. A. Stonehill in U.S. Pat. No. 3,282,775 (Nov. 1966). This inventor, however, referred only to the use of cationic agents. Several examples were given in the A. A. Stonehill patent. They all pertained to chemical compositions using glutaraldehyde solutions with quaternary ammonium salts or cetylpyridinum chloride both of which exhibited sporicidal characteristics at room temperature within the 4 to 9 pH range.
It is an object of the present invention to show that a glutaraldehyde solution combined with nonionic or anionic agents such as ethoxylates of isomeric linear alcohols or alkyl aryl sulfonates is far more active than any other previously known sporicidal formula based upon the mixing of glutaraldehyde with cationic agents.
It is a further object of the present invention to show that the combined use of glutaraldehyde solutions with nonionic or anionic surfactants is effective over a wider pH range (1 to 9) while also working at any temperature inside the 15.degree. to 75.degree.C range.
It is a further object of this invention to show that one can considerably reduce the sterilization time through simultaneous sonic or ultrasonic irradiation of the sporicidal compositions based upon a mixture of glutaraldehyde with nonionic or anionic surfactants.
To aid in the understanding of our invention, we shall briefly review the various physical or chemical mechanisms which play a role in the strong sporicidal effects observed in the method object of the present invention.
A few bacteria have evolved a highly effective mechanism for ensuring their survival; they exhibit an elementary form of differentiation in which, under certain conditions, the relatively sensitive vegetative form of the organism can give rise to a resistant dormant form, called a spore. Bacterial spores are much more resistant to adverse effects of heat, radiation and chemicals than their corresponding vegetative cells. The resistance of spores differs within the microbial population and species variation is common. Among the spores which were used to evaluate the methods object of the present invention, we shall mention Bacillus subtilis, Bacillus stearothermopilus, Bacillus pumilus, Clostridium sporogenes, and Clostridium tetani.
A bacterial spore is typically about one micro diameter and consists essentially of a small cell, often called the core of spore protoplast, surrounded by a number of specialized layers. The principal layers are the thick cortex and the multilayered coats and, around spores of certain species, a further loose and thin layer called exosporium.
At the moment it is believed (C. S. Phillips, Bact. Rev. 1962) that alkylating agents such as ethylene oxide, .beta. propiolactone, formaldehyde, glutaraldehyde as well as other aldehydes attack the sulfhydryl (--SH), hydroxyl (--OH), amino (--NH.sub.2) and carboxy ##EQU1## groups present in spore cell proteins. More recently T. J. Munton and A. D. Russell (J. Appl. Bact., 1970) stated that the chemical sites for glutaraldehyde action could involve --NH.sub.2 groups, including cross linking reactions between these groups (D. Hopwood, Histochemie, 1968). According to these authors, however, the suggested mechanism does not exclude sites of action with other chemical groups.
T. J. Munton and A. D. Russell (J. Appl. Bact. 1970) also showed that the uptake of acid glutaraldehyde and alkaline glutaraldehyde (sodium bicarbonate buffer) is similar and that both are of the Langmuirian type. This was demonstrated with E. Coli and Bacillus megatorium. In other words as more sites of the bacterial cell or spores are filled, glutaraldehyde molecules find increasing difficulty in attaching themselves to the cell or spore. In the methods object of the present invention it is believed that the nonionic linear alcohol ethoxylates decrease the surface tension and increase the wettability at the spore/liquid interface in such a manner that they promote a faster absorption rate of glutaraldehyde molecules. This could also be the result of the entrapping at the spore/liquid interface of a higher concentration of glutaraldehyde molecules, said phenomenon being increased in a logarithmic manner with temperature inside the 15.degree.-75.degree. range. Although of a lower magnitude, the same increased rate of absorption at the spore/liquid interface is observed with anionic alkyl aryl sulfonates mixed with nonionic polyoxyethylene alcohol ethers.
When speaking of absorption rates, one must point out that the increased wettability observed with the sporicidal molecules could be due not only to an increase at the external spore interface but also to a faster penetration inside the internal spore interfaces, i.e., across cortex layers, cortex or plasma membrane.
If using one of the sporicidal compositions object of the present invention in combination with ultrasonic irradiation extremely high killing rates are observed. This indeed could be explained in the following manner. As well known, the major component of a spore cortex layer is a polymer called murein (or peptidoglycan). Murein is present in lesser amounts in the walls of all bacteria. It is a large, cross-linked, net-like molecule exhibiting several unusual features. This polymer is acidic, and in spores may exist as a layer tightly contracted by some positively charged molecules. One recent theory to account for the extreme heat resistance of spores supposes that contractile pressure exerted by this structure may squeeze the central core sufficiently to maintain it in a state so dry as to confer heat resistance. Ultrasonic irradiation is one of the most efficient techniques (KY Sergeeva, Sov. Phys. Acoust., March 1966) to shake up polymer lattices and produce a fast depolymerization. This technique is said to be quite efficient over a wide frequency range both at low (G. Schmid et al., Kolloid L, 1951) and high frequency (M. A. K. Mostafa, J. Polym. Sci. 1958). It is, therefore, understandable that murein depolymerization or a partial destruction of the tight cross-linked lattice would enable the aldehyde groups to penetrate and combine faster with the active spore sites. Nonionic and anionic surfactants will indeed accelerate the penetration through the loosened polymer lattice. High intensity ultrasonic energy could also play an important role through other secondary but important mechanisms.
The proteinaceous outer coats of spores contain a disulfide-rich protein with some properties close to those of keratins. Since keratin-like proteins are typically strong, inert towards chemical reagents and resistant to enzymes they constitute an ideal protective barrier for spores. High intensity ultrasonics, however, could physically degrade keratin (J. H. Bradbury, Nature, 1960) and thus promote a faster penetration of active glutaraldehyde molecules.
Two more components characteristic of spores are high levels of calcium (often two percent of the spore's dry weight) and dipicolinic acid (DPA) which may account for over 10 percent of a spore's dry weight. Under acoustic turbulence ions exchange (Ca depletion) can take place while the heterocyclin DPA molecule could also be broken (I. E. Elpiner and A. V. Sokolskaya, Sov. Phys. Acoust. March 1963). In short, ultrasonic energy could either accelerate the physical diffusion of molecules or active radicals to reaction sites inside the spores, produce chemical bonds breakages of critical spores components (including sites modification) or both. It could also, specially with alkaline glutaraldehyde, depolymerize some of the glutaraldehyde in solution. This could be of particular significance when one remembers that alkalinized glutaraldehyde gradually loses its activity when polymerization progresses. (A. A. Stonehill et al, Am. Journ. Hosp. Phar. 1963).
Although the synergistic sporicidal effect due to a combination of moderate heat, glutaraldehyde solution and high intensity ultrasonics has been described already in G. Sierra's patent (Canadian Patent Application, No. 098,416, 1971), the present invention shows that an addition of nonionic or anionic surfactants to the glutaraldehyde solution leads in all cases to a substantial increase in bacteria, virus, or spore killing rate.